摘要
目的探讨β-氨基丙腈(β-APN)通过抑制赖氨酰氧化酶(LOX)减弱低氧诱导的非小细胞肺癌(NSCLC)转移侵袭的作用及其机制。方法低氧状态对NSCLC细胞LOX表达及催化活性的实验:将A549、SPCA1细胞系各分为常氧组、低氧12 h组、低氧24 h组;β-APN介导LOX失活导致NSCLC细胞迁移侵袭能力及上皮—间质转化(EMT)相关分子表达变化的实验:将A549、SPCA1细胞系分常氧组、低氧组、低氧+β-APN组。运用荧光法检测2种细胞LOX活性,采用逆转录及实时荧光定量PCR(qRT-PCR)检测细胞中低氧诱导因子-1α(HIF-1α)、LOX、上皮性钙黏附蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、基质金属蛋白酶2(MMP-2)、MMP-9基因表达情况,运用Western blot检测E-cadherin、N-cadherin、MMP-2、MMP-9蛋白水平。利用Transwell试验检测NSCLC细胞的转移侵袭能力。结果与常氧组比较,低氧12 h组和低氧24 h组A549及SPCA1细胞HIF-1α、LOX mRNA表达水平显著上调(P<0.01),HIF-1α、LOX蛋白水平显著上调(P<0.01),LOX酶活性显著增强(P<0.01)。与常氧组比较,低氧组A549及SPCA1细胞系的迁移能力和侵袭能力增强;与低氧组比较,低氧+β-APN组均显著减弱(P<0.01)。与常氧组比较,低氧组中E-cadherin mRNA的表达水平下降,N-cadherin、MMP-2及MMP-9 mRNA的表达水平上升;与低氧组比较,低氧+β-APN组E-cadherin mRNA的表达水平上升,N-cadherin、MMP-2及MMP-9 mRNA的表达水平下降(P<0.01)。与常氧组比较,低氧组E-cadherin蛋白水平下降及N-cadherin、MMP-2、MMP-9蛋白水平上升;与低氧组比较,低氧+β-APN组E-cadherin的蛋白水平上升,N-cadherin、MMP-2、MMP-9蛋白水平下降(P<0.01)。结论β-APN可通过抑制LOX酶减弱低氧诱导的NSCLC转移侵袭。
Objective To investigate the effect and mechanism of aminopropionitrile(βAPN)on hypoxia induced metastasis and invasion of non-small cell lung cancer(NSCLC)by inhibiting lysyl oxidase(LOX).Methods Experiments on the expression and catalytic activity of LOX in NSCLC cells under hypoxia state:A549 and SPCA1 cell lines were divided into normal oxygen group,hypoxia 12h group and hypoxia 24h group.Experiments on changes of NSCLC cell migration and invasion ability and epithelial mesenchymal transition(EMT)related molecular expression induced byβAPN mediated LOX inactivation:A549 and SPCA1 cell lines were divided into normal oxygen group,hypoxia group,hypoxia+βAPN group.LOX activity of the two cells was detected by fluorescence assay.The expression of hypoxia inducible factor-1α(HIF-1α),LOX,epithelial cadherin(E-cadherin),neural cadherin(N-cadherin),matrix metallopeptidase 2(MMP-2)and matrix metallopeptidase 9(MMP-9)genes were detected by reverse transcription and quantitative real time PCR(qRT PCR).The protein expression levels of E cadherin,N cadherin,MMP-2 and MMP-9 were detected by Western blot.Transwell assay was used to detect the migration and invasion ability of NSCLC cells.Results Compared with normorxia group,the expression levels of HIF-1αmRNA and LOX mRNA in A549 and SPCA1 cells in hypoxia 12h and hypoxia 24h group were significantly up regulated(P<0.01),HIF 1αprotein and LOX protein were significantly up-regulated(P<0.01),and LOX enzyme activity of were significantly increased(P<0.01).Compared with normorxia group,the migration and invasion ability of A549 and SPCA1 cell lines in hypoxia group were enhanced,while all significantly decreased in hypoxia+βAPN group(P<0.01).Compared with normorxia group,the expression level of E cadherin mRNA in hypoxia group was decreased,and the expression levels of N cadherin mRNA,MMP-2 mRNA and MMP-9 mRNA were increased,while the expression level of E cadherin mRNA increased,and the expression levels of N cadherin mRNA,MMP-2 mRNA and MMP-9 mRNA decreased in hypoxia+βAPN group(P<0.01).Compared with normorxia group,the expression of E cadherin protein decreased and the expression of N cadherin,MMP2 and MMP9 protein increased in the hypoxia group,while the expression levels of N cadherin,MMP-2 and MMP-9 protein decreased,E cadherin protein was increased in hypoxia+βAPN group(P<0.01).ConclusionβAPN can attenuate hypoxic induced migration and invasion of NSCLC by inhibiting LOX enzyme.
作者
王楷
王聪剑
鞠雪涛
焦振华
喻钧
Wang Kai;Wang Congjian;Ju Xuetao;Jiao Zhenhua;Yu Jun(Department of Thoracic Surgery,Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology,Hubei Province,Wuhan 430030,China)
出处
《疑难病杂志》
CAS
2024年第10期1159-1164,1170,共7页
Chinese Journal of Difficult and Complicated Cases
基金
国家自然科学基金(81902347)。
作者简介
通信作者:喻钧,E-mail:junyu2018@hotmail.com。
